This invention pertains to transgenic Brassica plants, plant material and seeds, characterized by harboring a specific transformation event, particularly by the presence of a male-sterility gene, at a specific location in the Brassica genome. The Brassica plants of the invention combine the male-sterility phenotype with optimal agronomic performance, genetic stability and adaptability to different genetic backgrounds.
All documents cited herein are hereby incorporated herein by reference.
The phenotypic expression of a transgene in a plant is determined both by the structure of the gene itself and by its location in the plant genome. At the same time the presence of the transgene (in a foreign DNA) at different locations in the genome will influence the overall phenotype of the plant in different ways. The agronomically or industrially successful introduction of a commercially interesting trait in a plant by genetic manipulation can be a lengthy procedure dependent on different factors. The actual transformation and regeneration of genetically transformed plants are only the first in a series of selection steps which include extensive genetic characterization, breeding, and evaluation in field trials.
The term xe2x80x9crapeseedxe2x80x9d covers every seed of the Brassica species. Brassica is cultivated from China and India to Finland and Canada as one of the most valuable oil crops. Most Brassica types belong to the family of Cruciferae. They originated as a diploid species having aneuploid chromosome numbers ranging from 7 (Brassica fruticulosa) to 12 (Sinapsis alba). The most extensively grown Brassica species in Canada is known as turnip rape, or Brassica campestris (aa, n=10). Brassica oleracea (cc, n=9) has diversified in recent evolutionary history into at least six major horticultural types, including broccoli, cauliflower and cabbage. Brassica nigra (bb, n=8) or black mustard is a less important crop commercially and is mainly known for its seeds from which mustard was originally made. From these basic types, amphiploid hybrids have been derived in more recent evolutionary stages by intercrossing. The most important of these are Brassica napus (aacc), of which the winter types provide the highest rapeseed yields in northern Europe and Brassica juncea (aabb) or brown mustard which is one of the major oil crops of the Indian sub-continent. Though intercrossing between different Brassica species (particularly those with compatible genomes) is possible and often done for breeding purposes, not all traits (or genes) will be able to be transferred from one species to the other or, when transferred to a different species, will retain identical characteristics (or expression patterns). Thus, a genetic locus conferring optimal expression of a natural or chimeric gene in one Brassica species, will not necessarily have the same effect in another.
Brassica species are bisexual and typically 60-70% self pollinated. The production of hybrids and introduction of genetic variation as a basis for selection was traditionally dependent on the adaptation of natural occurring phenomena such as self-incompatibility and cytoplasmic male sterility. Artificial pollination control methods such as manual emasculation or the use of gametocides are not widely applied in Brassica breeding due to their limited practicability and high cost respectively.
Transgenic methods have been developed for the production of male or female-sterile plants, which provide interesting alternatives to the traditional techniques. EP 0,344,029 describes a system for obtaining nuclear male sterility whereby plants are transformed with a male-sterility gene, which comprises for example a DNA encoding a barnase molecule under the control of a tapetum specific promoter TA29, which when incorporated into a plant ensures selective destruction of taptetum cells. Transformation of tobacco and oilseed rape plants with such a gene resulted in plants in which pollen formation was completely prevented (Mariani et al. 1990, Nature 347: 737-741). Cytochemical and histochemical analysis of anther development of Brassica napus plants comprising the chimeric pTA29:barnase gene is described by De Block and De Brouwer (1993, Planta 189:218-225).
To restore fertility in the progeny of a male-sterile plant, a system was developed whereby the male-sterile plant is crossed with a transgenic plant carrying a fertility-restorer gene, which when expressed is capable of inhibiting or preventing the activity of the male-sterility gene (U.S. Pat. Nos. 5,689,041; 5,792,929; De Block and De Brouwer, supra). The use of coregulating genes in the production of male-sterile plants to increase the frequency of transformants having good agronomical performance is described in WO96/26283. Typically, when the sterility DNA encodes a barnase, the coregulating DNA will encode a barstar.
Successful genetic transformation of Brassica species has been obtained by a number of methods including Agrobacterium infection (as described, for example in EP 0,116,718 and EP 0,270,882), microprojectile bombardment (as described, for example by Chen et al., 1994, Theor. Appl. Genet. 88:187-192) and direct DNA uptake into protoplasts (as described, for example by De Block et al. 1989, Plant Physiol. 914:694-701; Poulsen, 1996, Plant Breeding 115:209-225).
However, the foregoing documents fail to teach or suggest the present invention.
The invention relates to a transgenic Brassica plant, the genomic DNA of which is characterized by one or both of the following characteristics:
a) the genomic DNA is capable of yielding at least two, preferably at least three, more preferably at least four, most preferably five of the restriction fragments or sets of restriction fragments selected from the group of:
i) One set of NcoI fragments, one with a length of between 5077 and 14057 bp, preferably of about 6000 bp, and one with a length of between 2450 and 2838 bp, preferably of about 2500 bp;
ii) one set of EcoRV fragments wherein one has a length of between 5077 and 14057 bp, preferably of about 5500 bp and one with a length of between 4507 and 5077 bp, preferably of about 4800 bp;
iii) one set of MunI fragments, one with a length of between 5077 and 14057 bp, preferably with a length of about 5700 bp, and one with a length of between 2838 and 4799 bp, preferably of about 4500 bp;
iv) one HindIII fragment, with a length of between 2838 and 4507 bp, preferably with a length of about 3938 bp,
v) one EcoRI fragment, with a length of between 1989 and 2450 bp, preferably of about 2262 bp;
wherein each of the restriction fragments is capable of hybridizing under standard stringency conditions, with the +/xe2x88x922000 bp fragment obtainable by PCR amplification of a fragment of SEQ ID No. 1, using the probes having SEQ ID No. 2 and SEQ ID No. 3 and/or
b) the genomic DNA can be used to amplify a DNA fragment of between 160 and 200 bp, preferably of about 183 bp, using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID No.11 and SEQ ID No.12 respectively.
The present invention further relates to a transgenic Brassica plant, or seed, cells or tissues thereof, the genomic DNA of which is characterized in that it is capable of yielding at least two, preferably at least three, for instance at least four, more preferably five of the sets of restriction fragments selected from the group described under a) above comprising the sets of restriction fragments described under a) i), ii), iii), iv), and v) above, whereby the selection can include any combination of i), ii), iii), iv), and v) described under a) above.
The present invention further relates to a transgenic Brassica plant, or seed, cells, tissues or progeny thereof, the genomic DNA of which is characterized by both the characteristics described under a) and b) above.
The present invention further relates to a transgenic male-sterile Brassica plant, the genomic DNA of which is characterized by one, preferably by both the characteristics described under a) and b) above.
The invention also relates to the seed deposited at the ATCC, under accession number PTA-850, which will grow into a male sterile, herbicide resistant plant. The seed of ATCC deposit number PTA-850 comprises about 50% seed comprising the elite event of the invention, which will grow into male sterile, PPT tolerant plants. The seed can be sown and the growing plants can be treated with PPT or Liberty(trademark) as described herein, to obtain 100% male-sterile PPT tolerant plants comprising the elite event of the invention. The invention further relates to cells, tissues, progeny and descendants from a plant comprising the elite event of the invention grown from the seed deposited at the ATCC having accession number PTA-850. The invention further relates to plants obtainable by propagation of and/or breeding with a Brassica plant comprising the elite event of the invention grown from the seed deposited at the ATCC having accession number PTA-850.
The invention further relates to plants, seeds, cells or tissues comprising a foreign DNA sequence, preferably a male-sterility gene as described herein, integrated into the chromosomal DNA in a region which comprises the plant DNA sequence of SEQ ID No. 8 and/or SEQ ID No. 10, or a sequence which has at least 85% sequence identity to a sequence comprising the plant DNA sequence of SEQ ID No. 8 and/or SEQ ID No. 10.
The invention further provides a process for producing a transgenic cell of a Brassica plant, which comprises inserting a recombinant DNA molecule into a region of the chromosomal DNA of a Brassica cell which comprises the plant DNA sequence of SEQ ID No. 8 and/or SEQ ID No. 10, or a sequence which has at least 85% sequence identity with a sequence comprising the plant DNA sequence of SEQ ID No. 8 and/or SEQ ID No. 10, and, optionally, regenerating a Brassica plant from the transformed Brassica cell. 
The invention further relates to a method for identifying a transgenic plant, or cells or tissues thereof, which method comprises establishing one or both of the following characteristics of the genomic DNA of the transgenic plant, or its cells or tissues:
a) the genomic DNA is capable of yielding at least two, preferably at least three, more preferably at least four, most preferably five of the restriction fragments or sets of restriction fragments selected from the group of:
i) One set of NcoI fragments, one with a length of between 5077 and 14057 bp, preferably of about 6000 bp, and one with a length of between 2450 and 2838 bp, preferably of about 2500 bp;
ii) one set of EcoRV fragments wherein one has a length of between 5077 and 14057 bp, preferably of about 5500 bp and one with a length of between 4507 and 5077 bp, preferably of about 4800 bp;
iii) one set of MunI fragments, one with a length of between 5077 and 14057 bp, preferably with a length of about 5700 bp, and one with a length of between 2838 and 4799 bp, preferably of about 4500 bp;
iv) one HindIII fragment, with a length of between 2838 and 4507 bp, preferably with a length of about 3938 bp,
v) one EcoRI fragment, with a length of between 1989 and 2450 bp, preferably of about 2262 bp;
wherein each of the restriction fragments is capable of hybridizing under standard stringency conditions, with the +/xe2x88x922000 bp fragment obtainable by PCR amplification of a fragment of SEQ ID No. 1, using the probes having SEQ ID No. 2 and SEQ ID No. 3 and/or
b) the genomic DNA can be used to amplify a DNA fragment of between 160 and 200 bp, preferably of about 183 bp, using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID No.11 and SEQ ID No.12 respectively.
The invention further relates to a kit for identifying the transgenic plants comprising the elite event of the present invention, which kit comprises at least two PCR probes, one of which recognizes a sequence within the T-DNA of the elite event, the other recognizing a sequence within the 5xe2x80x2 or 3xe2x80x2 border flanking region of the elite event of the invention, preferably the PCR primers having the nucleotide sequence of SEQ ID No. 11 and SEQ ID No. 12, respectively, for use in the PCR identification protocol.